CN109187799A - A kind of fingerprint and its standard finger-print of perilla seed standard decoction - Google Patents
A kind of fingerprint and its standard finger-print of perilla seed standard decoction Download PDFInfo
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Abstract
The invention discloses a kind of detection methods of perilla seed standard decoction, it is established using high performance liquid chromatography, includes the following steps: that (1) prepares perilla seed test solution；(2) perilla seed control medicinal material solution and reference solution are prepared；(3) test solution to be measured is taken, is detected using high performance liquid chromatography, reference fingerprint is established.11 characteristic peaks are shared in the perilla seed standard decoction characteristic spectrum that the present invention establishes, the relative retention time specified value of each characteristic peak and the peak S are as follows: 0.340 (peak 1), 0.381 (peak 2), 0.457 (peak 3), 0.543 (peak 4), 0.684 (peak 5), 0.715 (peak 6), 0.936 (peak 7), 1.000 (peaks 8, S), 1.381 (peaks 9), 1.520 (peaks 10), 1.829 (peaks 11).This method is easy to operate, quickly, reproducible, can be used for the foundation of perilla seed standard decoction finger-print.
Description
Technical field
The invention belongs to Analysis of Chinese Traditional Medicine technical fields, and in particular to a kind of finger-print foundation side of perilla seed standard decoction
Method and its standard finger-print.
Background technique
Perilla seed is the dry mature fruit of Lamiaceae plant purple perilla Perilla frutescens (L.) Britt., is recorded
In the Pharmacopoeia of the People's Republic of China 2015 editions one, have effects that lowering the adverse-rising QI to resolve phlegm, relieving cough and asthma, relax bowel and defecation.Modern medicine
Pharmacological research shows that perilla seed mainly contains the ingredients such as fat oil, alkaloids, carbohydrate, flavonoids, has reducing blood lipid, cough-relieving
Relieving asthma, antiallergy, anti-aging, it is anti-oxidant the effects of.Flavonoids, phenolic acid class play important work in antibiosis in perilla seed
With for its drug system ultimate constituent.In terms of quality control, the beautiful China of paddy etc. is confused in qualitative, quantitative research perilla seed to change
On the basis of fragrant acid, thin-layer qualitative analysis has been carried out to its flavones ingredient luteolin, apiolin；In addition, there is document report pair
The content of fatty acid ingredient such as alpha-linolenic acid, linoleic acid or triterpenes components such as ursolic acid, oleanolic acid carries out in perilla seed
Measurement；2015 editions identifications for only having recorded perilla seed control medicinal material of the Pharmacopoeia of the People's Republic of China and Rosmarinic acid contain
It is fixed to measure.Due to the complicated component of Chinese medicine, effective component mostly changes with changing situations such as the place of production, source, collecting season again, because
This, the global feature of medicinal material is reflected using fingerprint pattern technology comprehensively, and further associated with its activity, current for improving
Method of quality control, guarantee clinical application is safely and effectively very necessary.Tang Xueyang etc. uses established HPLC-
PDA method characterizes perilla seed drug system characteristic spectrum quality, quantity and ancillary chemical type based on characteristic peak in characteristic spectrum
The characterization that 15 batches of perilla seed medicine materical crude slice are carried out with matter, based on characteristic index ingredient luteolin, apiolin and fan in characteristic spectrum
Repeatedly fragrant sour content and flavonoids (being characterized with luteolin), the content of phenolic acid class (being characterized with Rosmarinic acid) are to 15 batches of perilla seeds
The characterization of the medicine materical crude slice amount of progress, and based on benchmark medicine materical crude slice by the characterization result of 15 batches of perilla seed medicine materical crude slice quality and quantities being associated property respectively
Perilla seed prepared slice quality is effectively accurately evaluated in analysis.
According to " control of Chinese medicinal granule quality and the standard formulation technical requirements of Chinese Pharmacopoeia Commission's publication in 2016
(exposure draft) ", Chinese medicine standard decoction system follows traditional Chinese medical theory, is advised using qualified medicine materical crude slice according to clinical decoction decocting method
Generalized decocts, and is separated by solid-liquid separation, and is suitably concentrated and is made or is made through proper method is dry, as whether measuring Chinese medicinal granule
The almost the same marker with clinical decoction.Therefore the present invention establishes purple perilla substandard soup on forefathers' Research foundation
Agent characteristic spectrum and its content assaying method.Using perilla seed characteristic spectrum determined Rosmarinic acid, apiolin, luteolin,
11 characteristic peaks such as galuteolin, caffeic acid, wherein identifying 5 peaks.
Summary of the invention
The present invention provides the fingerprints and its standard finger-print of a kind of perilla seed standard decoction.
The present invention provides a kind of detection methods of perilla seed standard decoction, it is to use high performance liquid chromatography into foundation
, operating procedure is as follows:
(1) preparation of test solution: taking measuring samples, adds methanol solution ultrasonic extraction, filtering, take subsequent filtrate to get；
(2) Rosmarinic acid, apiolin, luteolin, coffee the preparation of reference solution: the preparation of reference substance solution: are taken
Acid, galuteolin reference substance, add methanol to dissolve；The preparation of control medicinal material solution: taking perilla seed control medicinal material, adds methanol solution super
Sound extract, filtration, take subsequent filtrate to get.
(3) reference substance solution is drawn respectively and test solution injects liquid chromatograph, chromatographic condition are as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；Mobile phase: using 0.1% formic acid as mobile phase A, with
Acetonitrile is Mobile phase B gradient elution；Flow velocity is 0.8-1.2mL/min；Column temperature: 20 DEG C~40 DEG C；Detection wavelength: 284nm；Gradient
Elution program is as follows:
Further, concentration of methanol solution described in step (1) is 80%；The ultrasonic extraction power is 600W, is mentioned
Taking frequency is 40kHz, extraction time 20min.
Further, the concentration of Rosmarinic acid, apiolin, luteolin, caffeic acid, galuteolin described in step (2)
Contain the solution of 80 μ g, 100 μ g, 100 μ g, 60 μ g, 60 μ g respectively for every 1ml.
Further, concentration of methanol solution is 80% in control medicinal material solution preparation method described in step (2)；Medicinal material with
Extraction solvent ratio is 1:25；The ultrasonic extraction power is 600W, and extraction frequency is 40kHz, extraction time 30min.
Further, it is 250mm, internal diameter that octadecylsilane chemically bonded silica described in step (3), which is the column length of filler,
For 4.6mm, granularity is 5 μm；Column temperature is 30 DEG C；Flow velocity is 1.0ml/min.
The present invention also provides a kind of discrimination methods of perilla seed standard decoction, it includes the following steps:
(1) perilla seed standard decoction extract is taken to prepare test solution；
(2) high performance liquid chromatography detection is carried out by the fingerprint of aforementioned perilla seed standard decoction；
(3) analysis detection result.
In a kind of standard finger-print of perilla seed standard decoction of the invention, in perilla seed standard decoction characteristic spectrum altogether
There are 11 characteristic peaks, the relative retention time specified value of each characteristic peak and the peak S are as follows: 0.340 (peak 1), 0.381 (peak 2), 0.457
(peak 3), 0.543 (peak 4), 0.684 (peak 5), 0.715 (peak 6), 0.936 (peak 7), 1.000 (peaks 8, S), 1.381 (peaks 9),
1.520 (peaks 10), 1.829 (peaks 11).
The present invention establishes perilla seed standard decoction characteristic spectrum and its content assaying method.Using perilla seed characteristic spectrum
11 characteristic peaks such as Rosmarinic acid, apiolin, luteolin, galuteolin, caffeic acid have been determined, and have identified therein 5
A characteristic peak.Whether almost the same with clinical decoction it can be used for measuring perilla seed granule.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is caffeic acid uv absorption spectra
Fig. 2 is galuteolin uv absorption spectra
Fig. 3 is Rosmarinic acid uv absorption spectra
Fig. 4 is luteolin uv absorption spectra
Fig. 5 is apiolin uv absorption spectra
Fig. 6 is that perilla seed standard decoction all band scans 3D map
Fig. 7 is that each wavelength of perilla seed standard decoction compares figure
Fig. 8 is the perilla seed decoction chromatogram under different column temperatures are investigated
Fig. 9 is the perilla seed decoction chromatogram under investigation different in flow rate
Figure 10 is the perilla seed decoction chromatogram under retardance experiment is investigated
Figure 11 is the perilla seed decoction freeze-dried powder chromatogram under different solvents are investigated
Figure 12 is the perilla seed decoction freeze-dried powder chromatogram under Different Extraction Method is investigated
Figure 13 is the perilla seed decoction freeze-dried powder chromatogram under investigating different extraction times
Figure 14 is the perilla seed decoction freeze-dried powder chromatogram under Different solution dosage is investigated
Figure 15 is the characteristic spectrum that perilla seed standard decoction chromatographic peak is pointed out
Figure 16 is the perilla seed decoction freeze-dried powder chromatogram under different instruments are investigated
Figure 17 is that perilla seed standard decoction chromatographic column durability investigates chromatogram
Figure 18 is that (each map represents batch as S1:ZSZBT180701 to perilla seed standard decoction characteristic spectrum；S2:
ZSZBT180702；S3:ZSZBT180703；S4:ZSZBT180704；S5:ZSZBT180705；S6:ZSZBT180706；S7:
ZSZBT180707；S8:ZSZBT180708；S9:ZSZBT180709；S10:ZSZBT180710；S11:ZSZBT180711；
S12:ZSZBT180712；S13:ZSZBT180713；S14:ZSZBT180714；S15:ZSZBT180715；S16:
ZSZBT180716；S17:ZSZBT180717)
Figure 19 is perilla seed standard decoction comparative diagram spectral peak (peak 3: caffeic acid；Peak 5: galuteolin；Peak 8 (S): rosemary
Acid；Peak 9: luteolin；Peak 10: apiolin)
Specific embodiment
The investigation of 1 perilla seed standard decoction chromatographic condition of embodiment
1, chromatographic condition and system suitability
(1) chromatographic condition drafted: using octadecylsilane chemically bonded silica as filler, (column length 250mm, internal diameter are
4.6mm, granularity are 5 μm)；Using 0.1% formic acid as mobile phase A, using acetonitrile as Mobile phase B, the regulation according to the form below carries out gradient
Elution；Flow velocity is 1.0mL/min；Column temperature is 30 DEG C；Detection wavelength is 284nm.Number of theoretical plate should not by the calculating of Rosmarinic acid peak
Lower than 3000.
(2) preparation of reference solution: taking Rosmarinic acid reference substance appropriate, adds methanol that the solution that 1mL contains 80 μ g is made.It takes
Apiolin, luteolin reference substance are appropriate, add methanol that the solution that every 1mL contains 100 μ g is respectively prepared.Take caffeic acid, reseda
Glycosides reference substance is appropriate, adds methanol that the solution that 1mL respectively contains 60 μ g is made.
(3) preparation of control medicinal material solution: taking perilla seed control medicinal material 1g, accurately weighed, sets in stuffed conical flask, accurate
80% methanol 25mL, close plug is added, weighed weight is ultrasonically treated (power 600W, frequency 40kHz) 30min, lets cool, then weighed
Weight is supplied the weight of less loss with 80% methanol, is shaken up, filtration, take subsequent filtrate to get.
(4) preparation of test solution: taking this product powder 0.5g, accurately weighed, sets in stuffed conical flask, and precision is added
80% methanol 50mL, close plug, weighed weight are ultrasonically treated (power 600W, frequency 40kHz) 20min, let cool, then weighed weight,
The weight that less loss is supplied with 80% methanol, shakes up, filtration, take subsequent filtrate to get.
(5) measuring method: precision draw 10 μ L of test solution, inject liquid chromatograph, measurement to get.
2, chromatographic condition is investigated
(1) selection of Detection wavelength: on the basis of the experiment condition drafted, using diode array detector respectively to coffee
Coffee acid, galuteolin, Rosmarinic acid, luteolin, apiolin are analyzed, and carry out all band scanning to test solution, and
Extract chromatogram of the test solution under 284nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm wavelength.It is each right
Attached drawing 1-5 is seen according to the uv absorption spectra of product, and perilla seed standard decoction all-wave length 3D scanning figure builds attached drawing 6, purple perilla substandard
Each wavelength of decoction compares figure and sees attached drawing 7.
The result shows that chromatographic peak information content is larger when Detection wavelength is 284nm, chromatogram baseline is more stable, each peak peak
Area is moderate, therefore Detection wavelength is determined as 284nm.
(2) it the investigation of column temperature: is investigated when being respectively 25 DEG C, 30 DEG C, 35 DEG C to column temperature.Chromatogram is shown in attached drawing 8, as a result
It is shown in Table 1.
1 column temperature investigation of table-characteristic peak relative retention time ratio
The result shows that when column temperature is respectively 25 DEG C, 30 DEG C, 35 DEG C, the relative retention time RSD of each characteristic peak is
0.00%-5.20%, when column temperature is 30 DEG C, chromatogram peak shape is preferable, and separating degree is moderate, therefore column temperature is determined as 30 DEG C.
(3) flow velocity is investigated: on the basis of the experiment condition drafted, be respectively 0.8mL/min, 1.0mL/min to flow velocity,
It is investigated when 1.2mL/min.The results are shown in attached figure 9, table 2.
2 flow velocity investigation of table-characteristic peak relative retention time ratio
The result shows that when flow velocity is respectively 0.8mL/min, 1.0mL/min, 1.2mL/min, the opposite reservation of each characteristic peak
Time RSD is 0.00%~7.25%, and when flow velocity is 1.0mL/min, chromatogram peak shape is preferable, and separating degree is moderate.Therefore flow velocity
It is determined as 1.0mL/min.
(4) retardance is tested: on the basis of the experiment condition drafted, chromatogram acquisition time being extended to 120min.Knot
Fruit sees attached drawing 10.The result shows that after chromatogram collects 60min, when chromatographic peak being acquired completely, therefore chromatogram being acquired
Between be determined as 60min.
In conclusion perilla seed standard decoction characteristic spectrum chromatographic condition and system suitability test determine are as follows: with 18
Alkyl silane bonded silica gel is filler (column length 250mm, internal diameter 4.6mm, granularity are 5 μm)；It is flowing with 0.1% formic acid
Phase A, using acetonitrile as Mobile phase B, the regulation according to the form below carries out gradient elution；Flow velocity is 1.0mL/min；Column temperature is 30 DEG C；Inspection
Survey wavelength is 284nm.Number of theoretical plate is calculated by Rosmarinic acid peak should be not less than 3000.The chromatographic condition of mobile phase is shown in Table 3.
The 3 final eluent gradient of perilla seed standard decoction characteristic spectrum of table
The sample solution preparation method of 2 perilla seed standard decoction of embodiment is investigated
1, the investigation of Extraction solvent: taking freeze-dried powder (lot number: ZSZBT180701) 0.5g of perilla seed standard decoction, accurate
It is weighed, it sets in stuffed conical flask, is investigated when being respectively methanol, 80% methanol, water, ethyl alcohol to test sample Extraction solvent, it is close
Plug, weighed weight are ultrasonically treated (power 600W, frequency 40kHz) 30min, let cool, then weighed weight, supplied with Extraction solvent
The weight of less loss, shakes up, filtration, take subsequent filtrate to get.The results are shown in attached figure 11.
The result shows that resulting chromatogram is close, and chromatographic peak contains much information when mobile phase is methanol and 80% methanol,
Separating degree is good.Consider rosmarinic acid contents measurement, therefore selects 80% methanol as Extraction solvent.
2, extracting method is investigated: taking freeze-dried powder (lot number: ZSZBT180701) 0.5g of perilla seed standard decoction, precision claims
It is fixed, it sets in stuffed conical flask, 80% methanol 50mL, close plug is added in precision, and weighed weight is respectively back test sample extracting method
It is investigated when stream, ultrasound, extraction time 30min is let cool, then weighed weight, and the weight of less loss is supplied with 80% methanol, is shaken
It is even, filtration, take subsequent filtrate to get.The results are shown in attached figure 12.
The result shows that it is consistent with effect when refluxing extraction to carry out ultrasonic extraction respectively to test sample.Because ultrasonic extraction operates
It is more easy, therefore test sample extracting method is determined as ultrasonic extraction.
3, extraction time is investigated: taking freeze-dried powder (lot number: ZSZBT180701) 0.5g of perilla seed standard decoction, precision claims
It is fixed, it sets in stuffed conical flask, 80% methanol 50mL, close plug is added in precision, and weighed weight is ultrasonically treated (power 600W, frequency
40kHz), it respectively to test sample extraction time to be investigated when 20min, 30min, 40min, lets cool, then weighed weight, uses
80% methanol supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.The results are shown in attached figure 13.
The result shows that can sufficiently be extracted when being 20min between at the extraction.Therefore test sample extraction time is determined as
20min。
4, solvent dosage is investigated: taking freeze-dried powder (lot number: ZSZBT180701) 0.5g of perilla seed standard decoction, precision claims
It is fixed, it sets in stuffed conical flask, 80% methanol, close plug is added in precision, and weighed weight is ultrasonically treated (power 600W, frequency 40kHz)
20min is respectively that 25mL, 50mL, 75mL are investigated to test sample solvent dosage, lets cool, then weighed weight, with 80% methanol
The weight for supplying less loss, shakes up, filtration, take subsequent filtrate to get.The results are shown in attached figure 14.
The result shows that chromatogram peak area is of moderate size when solvent dosage is 50mL.Therefore test sample solvent dosage determines
For 50mL.
The fingerprint of 3 perilla seed standard decoction of embodiment is investigated
1, chromatographic peak is pointed out
The preparation of solution: by the experiment condition drafted, perilla seed standard decoction test solution is prepared.
The preparation of reference solution: taking apiolin, luteolin reference substance appropriate, adds methanol that every 1mL is respectively prepared and contains
The solution of 100 μ g.It takes Rosmarinic acid reference substance appropriate, adds methanol that the solution that 1mL contains 80 μ g is made.Take caffeic acid, galuteolin
Appropriate reference substance adds methanol that the solution that 1mL respectively contains 60 μ g is made.
The preparation of control medicinal material solution: taking perilla seed control medicinal material 1g, accurately weighed, sets in stuffed conical flask, and precision adds
Enter 80% methanol 25mL, close plug, weighed weight is ultrasonically treated (power 600W, frequency 40kHz) 30 minutes, lets cool, then weighed heavy
Amount, the weight of less loss is supplied with 80% methanol, is shaken up, filter, take subsequent filtrate to get.
The preparation of negative control solution: it by the experiment condition drafted above, prepares and lacks perilla seed standard decoction negative control
Solution.Perilla seed standard decoction characteristic pattern spectral peak is positioned.The results are shown in attached figure 15.
The result shows that peak 3 is caffeic acid, peak 5 is galuteolin, peak 8 is Rosmarinic acid, peak 9 is luteolin, peak 10 is
Apiolin.In following methods investigation, 11 peaks in sample are investigated.
2, precision test: precision weighs 1 part of perilla seed standard decoction freeze-dried powder (lot number: ZSZBT180701), by drafting
It is prepared by experimental method, and continuous sample introduction 6 times.It the results are shown in Table 4, table 5.
4 precision retention time of table investigates result
5 precision peak area of table investigates result
The result shows that the RSD value of each peak retention time is 0.02%-0.16%, the RSD value of peak area is 0.09%-
4.05%.The instrument precision is good.
3, repeatability is investigated: precision weighs 6 parts of perilla seed standard decoction freeze-dried powder (lot number: ZSZBT180701), by drafting
Experimental method prepared and measured.It the results are shown in Table 6, table 7.
Repeated investigation-characteristic peak relative retention time the ratio of table 6
Repeated investigation-characteristic peak relative peak area the ratio of table 7
The result shows that the RSD of each characteristic peak relative retention time is 0.00%~0.36%, each characteristic peak relative peak area
RSD 0.00%~4.45%, this method is reproducible.
4, Intermediate precision is investigated
(1) different instruments are investigated: on the basis of the experiment condition drafted, precision weighs the freeze-drying of perilla seed standard decoction respectively
Two parts of powder (lot number: ZSZBT180701), prepares test solution, respectively on Agilent 1260, Waters e2695-2998, island
(chromatographic column is 250 × 4.6mm of Kromasil 5HC-C18) is measured on saliva LC-20AT high performance liquid chromatograph.See attached
Figure 16, table 8, table 9.
The different instrument investigation-characteristic peak relative retention time ratios of table 8
The different instrument investigation-characteristic peak relative peak area ratios of table 9
The result shows that the RSD of each characteristic peak relative retention time exists when being detected with above-mentioned 3 kinds of instruments to test sample
0.00%~7.75%；The RSD of each characteristic peak relative peak area is 0.00%~73.60%.
(3) different personnel and time are investigated: on the basis of the experiment condition drafted above, by different personnel (A, B) not
Precision weighs each two parts of perilla seed standard decoction freeze-dried powder (lot number: ZSZBT180701) to same time (T1, T2) respectively, and preparation supplies
Test product is measured.It is shown in Table 10, table 11.
The different personnel of table 10 and time investigation-characteristic peak relative retention time ratio
The different personnel of table 11 and time investigation-characteristic peak relative peak area ratio
The result shows that under different sample preparation personnel and different sample preparation time conditions, when each characteristic peak retains relatively
Between RSD 0.00%~0.18%；The RSD of each characteristic peak relative peak area is 0.00%~8.65%.
5, durability is investigated: being respectively Agilent ZORBAX to chromatographic column on the basis of the experiment condition drafted
Eclipse XDB-C18 (250 × 4.6mm, 5 μm), Waters C18 (5 μm of 250 × 4.6mm, 5 μm 4.6 of KromasilC18
× 250mm is investigated).The results are shown in attached figure 17, table 12, table 13.
12 chromatographic column durability investigation of table-characteristic peak relative retention time
13 chromatographic column durability investigation of table-characteristic peak relative peak area ratio
The result shows that being detected with above-mentioned 3 kinds of chromatographic columns to sample, the RSD of characteristic peak relative retention time is 0.00
The RSD of~4.76% peak relative peak area is 0.00%~12.41%.
6, study on the stability: on the basis of the experiment condition drafted, taking same test solution, respectively at 0h, 2h, 4h,
8h, 12h, for 24 hours when measure.It the results are shown in Table 14, table 15.
14 study on the stability of table-characteristic peak relative retention time
15 study on the stability of table-characteristic peak relative peak area
The result shows that the RSD of corresponding characteristic peak relative retention time is 0.00%~1.41%, characteristic peak is opposite
The RSD of peak area is 0.00%~7.98%.
In conclusion the RSD of each characteristic peak relative retention time meets the requirements in above every investigation, this method is good
It is good, it can be used for the research of perilla seed standard decoction finger-print.
4 perilla seed standard decoction standard finger-print of embodiment
1, the preparation of reference solution: taking Rosmarinic acid reference substance appropriate, adds methanol that the solution that 1mL contains 80 μ g is made.It takes
Apiolin, luteolin reference substance are appropriate, add methanol that the solution that every 1mL contains 100 μ g is respectively prepared.Take caffeic acid, reseda
Glycosides reference substance is appropriate, adds methanol that the solution that 1mL respectively contains 60 μ g is made.
2, the preparation of control medicinal material solution: taking perilla seed control medicinal material 1g, accurately weighed, sets in stuffed conical flask, accurate
80% methanol 25mL, close plug is added, weighed weight is ultrasonically treated (power 600W, frequency 40kHz) 30min, lets cool, then weighed
Weight is supplied the weight of less loss with 80% methanol, is shaken up, filtration, take subsequent filtrate to get.
3, the preparation of test solution: taking this product powder 0.5g, accurately weighed, sets in stuffed conical flask, and precision is added
80% methanol 50mL, close plug, weighed weight, ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes are let cool, then weighed heavy
Amount, the weight of less loss is supplied with 80% methanol, is shaken up, filter, take subsequent filtrate to get.
4, chromatographic condition: using octadecylsilane chemically bonded silica as filler (column length 250mm, internal diameter 4.6mm, grain
Degree is 5 μm)；Using 0.1% formic acid as mobile phase A, using acetonitrile as Mobile phase B, the regulation according to the form below carries out gradient elution；Flow velocity
For 1.0mL per minute；Column temperature is 30 DEG C；Detection wavelength is 284nm.Number of theoretical plate should be not less than by the calculating of Rosmarinic acid peak
3000。
5, measure: drawing reference substance solution and each 10 μ l of test solution respectively, inject liquid chromatograph, measurement to get.
6, standard finger-print is established: take 17 batches of perilla seed standard decoctions, prepare test solution, then by chromatographic condition into
Row measurement, record HPLC figure, using similarity evaluation (2012 editions) to 17 batches of purple perilla substandards
Decoction is synthesized, and the finger-print and control map of perilla seed standard decoction characteristic spectrum are established.The results are shown in attached figure 18-19,
Table 16-17.
16 17 batches of perilla seed standard decoction relative retention times of table
17 17 batches of perilla seed standard decoction relative peak areas of table
According to the principle that relative retention time stabilization and each batch sample can detect, select 11 repeatability preferable altogether
Peak is as characteristic peak.The result shows that when peak 8 is as the peak S, 17 batch perilla seed standard decoction characteristic peak relative peak area RSD
2.0% is respectively less than in 0.00%~59.92%, 17 batch perilla seed standard decoction, 11 characteristic peak relative retention time RSD.But
In different instruments and different chromatographic columns are investigated, the RSD of relative retention time is 0.00%~7.75%, therefore, fixes tentatively for examination
The relative retention time of product should be within ± the 10% of specified value.
Final regulation: should be presented 11 characteristic peaks in test sample characteristic spectrum, wherein when 1 peak should retain with object of reference peak
Between it is identical, peak corresponding with Rosmarinic acid object of reference is the peak S, calculates the relative retention time of each characteristic peak Yu the peak S, opposite guarantor
Stay the time should be within ± the 10% of specified value.Specified value are as follows: 0.340 (peak 1), 0.381 (peak 2), 0.457 (peak 3), 0.543
(peak 4), 0.684 (peak 5), 0.715 (peak 6), 0.936 (peak 7), 1.000 (peaks 8, S), 1.381 (peaks 9), 1.520 (peaks 10),
1.829 (peaks 11).
Claims (10)
1. a kind of detection method of perilla seed standard decoction, it is characterised in that: it is established using high performance liquid chromatography,
Operating procedure is as follows:
(1) preparation of test solution: taking measuring samples, adds methanol solution ultrasonic extraction, filtering, take subsequent filtrate to get；
(2) preparation of reference solution: Rosmarinic acid, apiolin, luteolin, caffeic acid, galuteolin reference substance are taken, first is added
Alcohol dissolution；The preparation of control medicinal material solution: taking perilla seed control medicinal material, adds methanol solution ultrasonic extraction, and filtration takes subsequent filtrate,
To obtain the final product.
(3) reference substance solution being drawn respectively and test solution injecting liquid chromatograph, chromatographic condition is as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；Mobile phase: using 0.1% formic acid as mobile phase A, with acetonitrile
For Mobile phase B gradient elution；Flow velocity is 0.8-1.2m/min；Column temperature: 25 DEG C~35 DEG C；Detection wavelength: 284nm；Gradient elution
Program is as follows:
2. detection method according to claim 1, it is characterised in that: concentration of methanol solution described in step (1) is 80%；
The ultrasonic extraction power is 600W, and extraction frequency is 40kHz, extraction time 20min.
3. detection method according to claim 1, it is characterised in that: Rosmarinic acid, apiolin, wood described in step (2)
Rhinoceros grass element, caffeic acid, galuteolin concentration contain the solution of 80 μ g, 100 μ g, 100 μ g, 60 μ g, 60 μ g respectively for every 1ml.
4. detection method according to claim 1, it is characterised in that: control medicinal material solution preparation side described in step (2)
Concentration of methanol solution is 80%. in method.
5. detection method according to claim 1, it is characterised in that: step (2) medicinal material and Extraction solvent ratio are
1:25。
6. detection method according to claim 1, it is characterised in that: ultrasonic extraction power described in step (2) is 600W,
Extraction frequency is 40kHz, extraction time 30min.
7. detection method according to claim 1, it is characterised in that: octadecylsilane bonded silica described in step (3)
Glue is that the column length of filler is 250mm, and internal diameter 4.6mm, granularity is 5 μm.
8. detection method according to claim 1, it is characterised in that: in step (3), column temperature is 30 DEG C；Flow velocity is
1.0mL/min。
9. a kind of discrimination method of perilla seed standard decoction, it is characterised in that: it includes the following steps:
(1) perilla seed standard decoction extract is taken to prepare test solution；
(2) by the detection of high performance liquid chromatography described in claim 1-8；
(3) analysis detection result.
10. discrimination method according to claim 9, it is characterised in that: share 11 in perilla seed standard decoction characteristic spectrum
A characteristic peak, the relative retention time specified value of each characteristic peak and the peak S are as follows: 0.340 (peak 1), 0.381 (peak 2), 0.457 (peak
3), 0.543 (peak 4), 0.684 (peak 5), 0.715 (peak 6), 0.936 (peak 7), 1.000 (peaks 8, S), 1.381 (peaks 9), 1.520
(peak 10), 1.829 (peaks 11).
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